Mitochondria play a central role in the control of apoptosis. Members of the Bcl-2 family regulate the release of mitochondrial factors including cytochrome c and Smac. Members of the subfamily sharing homology only in the BCL-2 homology domain 3 (BH3-only proteins, or BOPs) respond to cellular stress by translocating to the mitochondria, where they collaborate with multidomain Bcl-2 family members to achieve the release of cytochrome c and other intermembrane constituents. This process involves several steps: 1) translocation to the mitochondrial outer membrane; 2) dissociation of cytochrome c from inner membrane; 3) conformational change of Bax/Bak characterized by alkali-resistant insertion; and 4) transit of intermembrane space proteins through the outer membrane. We propose to characterize the mechanism by which BOPs achieve each of these steps, and will focus primarily on the molecular basis for cytochrome c dissociation from the inner membrane. Cardiolipin plays a critical role in cytochrome binding and mitochondrial membrane structure. In Aim I, we will test the hypothesis that BOPs cause a change in cardiolipin abundance, oxidation, distribution, or structure, leading to cytochrome c release. In Aim II, we will test the hypothesis that cytochrome c dissociation is due to an effect on its interaction with the electron transfer complexes, and will also assess the role of ceramide in this interaction. Finally, in Aim III, we will assess the effect of Bcl-2 and BcI-xL on the four steps involved in cytochrome c release mediated by BOP, in order to gain insight into the mechanisms by which these anti-apoptotic molecules protect the mitochondria. These studies will elucidate the molecular mechanism by which BH3-only proteins cause the release of cytochrome c from mitochondria. [unreadable] [unreadable]